Cleaning solution and application thereof in alkaline phosphatase magnetic particle chemiluminescence

Abstract

The invention provides a cleaning solution and application thereof in alkaline phosphatase magnetic particle chemiluminescence, the formula of the cleaning solution comprises optimized screening of a buffer system, metal ions, a surfactant, a preservative and a defoaming agent, the pH value is 8-9, and the adverse effect on protein in a to-be-detected sample is reduced while the alkaline phosphatase activity effect is met; through the synergistic effect of the nonionic surfactant and the zwitterionic surfactant, the zwitterionic surfactant is preferably C12imSP, and the nonionic surfactant is preferably C12imSP. The preservative is selected from an environment-friendly and safe Krovin series, the antibacterial spectrum is wide, the use limitation of a single component under the condition of a low-concentration preservative is broken through, and the stability of the cleaning fluid is further improved.

Description

Technical Field

[0001] The invention relates to the technical field of in vitro diagnostic reagents for medical devices, and in particular to a cleaning solution and application thereof in chemiluminescence of alkaline phosphatase magnetic particles. Background Art

[0002] Magnetic particle chemiluminescence is a combination of magnetic particle molecular technology, chemiluminescence technology and immunoassay technology. It uses the rapid and easy automation of magnetic separation technology, the high sensitivity of chemiluminescence technology and the specificity of immunoassay to detect various antigens, haptens, antibodies, etc., so it has the advantages of high automation, high sensitivity and strong specificity. Among them, magnetic particle chemiluminescence detection based on alkaline phosphatase is one of the commonly used systems. Alkaline phosphatase (ALP) is used as a marker enzyme to indirectly determine the content of the analyte by catalyzing the luminescence of specific substrates (such as 1,2-dioxetane derivatives AMPPD or 9-(4-chlorophenylthiophosphoryloxymethylene)-10-methyldihydroacridine disodium salt APS-5, etc.). The optimal Ph value of enzyme hydrolysis activity is about 10. In a suitable buffer, with the catalytic hydrolysis of the enzyme, the substrate is decomposed and emits a very high intensity light signal. The speed of its luminescence depends on the concentration of alkaline phosphatase, which makes the detection range of magnetic particle chemiluminescence wider, the stability is better and the luminescence efficiency is higher.

[0003] The factors that affect the performance of the alkaline phosphatase magnetic microparticle chemiluminescence detection system, in addition to the activity and stability of the conjugate composed of different detection objects, mainly lie in the control of the precision of the reaction system. Among them, the cleaning solution, as a necessary reagent component of the phosphatase magnetic microparticle chemiluminescence detection system, will be used in conjunction with various immunoassay kits to clean the trace substances remaining in the reaction cup and / or on the needle, and to reduce the interference of non-specific binding caused by trace substances, thereby further improving the performance indicators such as detection sensitivity, linear range, specificity and stability. Therefore, if an optimized and universal cleaning solution suitable for the alkaline phosphatase magnetic microparticle chemiluminescence detection system can be provided, it is of far-reaching significance to further improve the detection sensitivity while improving the cleaning ability.

[0004] Common cleaning solution formulas include buffer systems (pH 9-10), salt ions (Na, Mg), surfactants (sodium EDTA, Gemini, Tween-20 or Tween 80), stabilizers (proteins, polysaccharides, etc.), preservatives (PC300 / Proclin300, Bronidox or sodium azide) and defoaming agents (silicone oil type). However, these components have the following problems in the chemiluminescence detection of alkaline phosphatase magnetic particles: (1) pH 9-10 is the optimal working range of alkaline phosphatase. Too low pH (7-8) is not conducive to the effective activation of alkaline phosphatase, but too high pH (9-10) will also cause changes in the protein conformation of the test substance. Further affecting the accuracy of the test results; (2) Sodium EDTA will chelate the activator metal ions Mg and Zn to form a stable chelate, depriving these metal ions of their activation effect on alkaline phosphatase, resulting in structural changes in the enzyme active center and a decrease in catalytic function, making it unsuitable for coexistence; (3) Cationic surfactants (such as Gemini), whose hydrophilic groups carry a positive charge, can exhibit good surface activity in acidic media, but easily lose their surface activity in alkaline media with a pH>7; (4) Alkaline phosphatase is a zinc-containing metal enzyme. Zinc ions are easily detached from alkaline phosphatase by related chelation in the solution, thereby affecting the activity of the enzyme. Summary of the invention

[0005] In order to overcome the above problems, the present invention provides a universal blocking agent with relatively low pH value and stable performance and a blocking solution prepared by the blocking agent, which can be used in latex immunoturbidimetric detection method, especially in the preparation of reagent R2 in the preparation of latex reagent.

[0006] In order to achieve the above-mentioned invention object, the present invention is implemented by the following technical solutions:

[0007] In a first aspect, the present invention provides a cleaning solution for detecting the chemiluminescence of alkaline phosphatase magnetic microparticles, the cleaning solution comprising a buffer solution, metal ions, a surfactant and a preservative, with a pH value of 8 to 9; the buffer solution is a Tris buffer solution, and the working concentration is 50 to 200 mM; the metal ions at least include zinc ions and sodium ions, which are derived from zinc chloride and sodium chloride, respectively; the metal ions at least further include magnesium ions and / or calcium ions, the magnesium ions are derived from soluble salts containing magnesium ions, such as magnesium chloride and / or magnesium sulfate; the calcium ions are derived from soluble salts containing calcium ions, such as calcium chloride and / or calcium acetate; the surfactant comprises both a nonionic surfactant and a zwitterionic surfactant, wherein the zwitterionic surfactant is an imidazoline-type structure; and the preservative is a Krovin series preservative.

[0008] According to a preferred embodiment of the present invention, the pH value of the cleaning solution is 8-8.8.

[0009] Preferably, the pH value of the cleaning solution is 8.3.

[0010] According to a preferred embodiment of the present invention, the working concentration of zinc ions in the cleaning solution is 0.01-0.2 mM, the working concentration of sodium ions is 40 g / L-200 g / L; the working concentration of magnesium ions or calcium ions is 1-100 mM; the non-ionic surfactants are Tween 20 and Brij35, and the working concentrations are both 0.05%-1%; the working concentration of zwitterionic surfactants is 0.1%-1%; the working concentration of preservatives is 0.05%-1%; and a defoamer may also be included, which is a polyether defoamer with a working concentration of 0.01-0.1%.

[0011] Preferably, the working concentration of sodium ions is 100 g / L; the nonionic surfactants are Tween 20 and Brij35, both with working concentrations of 0.5%; the working concentration of zwitterionic surfactants is 0.5%; and the working concentration of defoaming agent is 0.1%.

[0012] According to a preferred embodiment of the present invention, the pH value of the cleaning solution is 8.3-8.8; the zwitterionic surfactant is 1-sulfopropyl-3-dodecyl imidazole inner salt, referred to as C 12 imSP.

[0013] According to a preferred embodiment of the present invention, the total working concentration of magnesium ions and / or calcium ions in the cleaning solution does not exceed 100 mM; the total mass concentration of non-ionic surfactants does not exceed 1.5%.

[0014] According to a preferred embodiment of the present invention, the Krovin series preservatives in the cleaning solution are Krovin100, Krovin300, and Krovin500, and any one of the Krovin series preservatives can be selected and used alone.

[0015] Preferably, the preservative is Krovin500.

[0016] According to a preferred embodiment of the present invention, C 12 imSP was prepared in-house using imidazole, acrylonitrile, 1-bromododecane and propane sultone as raw materials.

[0017] According to a preferred embodiment of the present invention, C 12The preparation method of imSP is as follows: dissolving imidazole in methanol at 40-60°C, adding acrylonitrile to react for 6-10 hours to obtain nitrile ethyl imidazole; reacting nitrile ethyl imidazole and 1-bromododecane in isopropanol solvent at 80°C for 16-20 hours, removing isopropanol by rotary evaporation, stirring the obtained residue with NaOH solution at a mass ratio of 10-20% and mixing to obtain a mixed solution; extracting the mixed solution with chloroform to obtain 1-dodecyl imidazole; placing 1-dodecyl imidazole in an ethanol / water solvent at 70-80°C and a pH value of 8-9, adding propane sultone to react for 6-10 hours, and the reaction product includes the target product C 12 imSP.

[0018] Preferably, the cleaning solution contains C 12 The preparation method of imSP is as follows: imidazole is dissolved in methanol at 50°C, acrylonitrile is added and reacted for 8 hours to obtain nitrile ethyl imidazole; nitrile ethyl imidazole and 1-bromododecane are reacted in isopropanol solvent at 80°C for 18 hours, isopropanol is removed by rotary evaporation, the obtained residue is mixed with NaOH solution at a mass ratio of 15% and stirred to obtain a mixed solution; the mixed solution is extracted with chloroform to obtain 1-dodecyl imidazole; 1-dodecyl imidazole is placed in an ethanol / water solvent at 75°C and a pH value of 8.3, propane sultone is added and reacted for 8 hours to obtain the target product C 12 imSP.

[0019] According to a preferred embodiment of the present invention, the working concentration in the cleaning solution formula is converted into a mother liquor concentrated 10 to 20 times for configuration or storage, and diluted to the working concentration with purified water when used.

[0020] Preferably, the working concentration in the cleaning solution formula is converted into a 20-fold concentrated mother solution for preparation or storage.

[0021] In the second aspect, the present invention provides a method for preparing a cleaning solution, wherein the working concentration in the cleaning solution formula is converted into a 10 to 20 times concentrated ratio and added to purified water, the pH value is adjusted after stirring evenly, and the solution is filtered with a 0.22 um filter membrane. The mother liquor of the cleaning solution is obtained after filtration and is stored at 4°C for later use after packaging.

[0022] According to a preferred embodiment of the present invention, the preparation is preferably performed at a concentration ratio of 20 times.

[0023] In a third aspect, the present invention provides a cleaning solution and a preparation method thereof, and application thereof in the preparation of products related to alkaline phosphatase chemiluminescence detection.

[0024] The beneficial effects of the present invention are:

[0025] The present invention provides a cleaning solution for detecting chemiluminescence of alkaline phosphatase magnetic microparticles. The formula of the cleaning solution includes the optimization and screening of a buffer system, metal ions, a surfactant, a preservative and a defoaming agent, and solves the problem that a higher pH value (9-10) is beneficial to activating the activity of alkaline phosphatase while causing the influence of protein conformation change. The present invention selects a slightly lower pH value (8-9) within the activity range of alkaline phosphatase, and reduces the adverse effect on the protein in the sample to be tested while satisfying the activity of alkaline phosphatase; through the synergistic effect of a nonionic surfactant and a zwitterionic surfactant, wherein the zwitterionic surfactant is preferably C 12 imSP; The environmentally friendly and safe Krovin series is preferred as the preservative, which has a wide antibacterial spectrum, breaks the limitation of using a single component under low concentration preservative conditions, and further improves the stability of the cleaning solution. And its application in alkaline phosphatase magnetic particle chemiluminescence.

[0026] Regarding the buffer, in order to maintain the activity of alkaline phosphatase, pH value is the most important trade-off indicator. Alkaline phosphatase can show higher activity at higher pH values. However, too high pH values ​​may not only cause changes in the conformation of proteins such as the test substance, but also lead to instability of the enzyme structure, thereby affecting the accuracy of the test results. The preferred zwitterionic buffer system of the present invention can overcome the inhibitory effect of phosphate on alkaline phosphatase in the phosphate buffer system, and the zwitterionic buffer system also has good pH buffering performance and higher stability. Common zwitterionic buffer systems include tris(hydroxymethyl)aminomethane (Tris), 3-cyclohexylaminopropanesulfonic acid (CAPS), 2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 3-(N-morpholino)propanesulfonic acid (MOPS), piperazine-N,N-bis(2-ethanesulfonic acid) (PIPES), piperazine-N,N-bis(2-hydroxyethanesulfonic acid) (POPSO), etc. In addition, the present invention preferably selects the Tris buffer system from the zwitterionic buffer system, which is easier to prepare, and Tris is used alone or in combination with other components. It has been verified that if the pH value is maintained in the range of 8.0 to 9.0, the relatively low pH buffer system is suitable for the active reaction of alkaline phosphatase and is conducive to maintaining the three-dimensional protein conformation structure of the test substance, etc., and the relatively low pH value can not only reduce the corrosiveness to the detection equipment and instruments and extend their service life, but also help to improve the sensitivity and accuracy of the results.

[0027] Although the pH value has only dropped by 1 unit, the activity of alkaline phosphatase can still be guaranteed within this lower range. In addition, under alkaline conditions, theoretically a higher pH value helps to maximize the elution of nonspecific antigen-antibody binding, but it will also weaken the interaction forces of certain specific bindings, resulting in weakened or destroyed detection sensitivity of specific binding. Reducing the pH by 1 unit is closer to ensuring the structure of common proteins, and is sufficient to make the chemiluminescent reaction system of specific binding more stable and more efficient. In scientific research, the reliability and repeatability of data are crucial. Although it has only been reduced by 1 pH unit, this change may be enough to affect the scope of use of common alkaline phosphatase chemiluminescent systems, improve the stability and reliability of experimental data, and further improve detection sensitivity.

[0028] Regarding the choice of metal ions, alkaline phosphatase is a dimeric protein that belongs to the category of zinc-containing metalloenzymes. Its structure contains three types of metal binding sites (usually labeled A, B, and C). Among these sites, the A site mainly binds zinc ions and is essential for maintaining the structure and catalytic activity of the enzyme. The B and C sites are able to bind other metal ions such as magnesium ions or calcium ions. When magnesium ions or calcium ions bind to the B and C sites, they can play an important role; specifically, the binding of metal ions can eliminate the interaction between the two subunits of alkaline phosphatase, allowing both subunits to be independently phosphorylated, and phosphorylation is a key step in activating the activity of alkaline phosphatase, which helps the enzyme catalyze the conversion of substrates in subsequent reactions. Therefore, in the cleaning solution used with alkaline phosphatase, metal ions such as zinc ions, magnesium ions, and calcium ions are usually included to maintain or activate the activity of alkaline phosphatase. Among them, magnesium ions usually come from soluble salts containing magnesium ions, such as magnesium chloride, magnesium sulfate, etc., which can dissociate magnesium ions in solution; calcium ions come from soluble salts containing calcium ions, such as calcium chloride, calcium acetate, etc. The addition of zinc ions is to prevent the loss of zinc ions at the A site of alkaline phosphatase due to chelation. The presence of these metal ions in the cleaning solution helps to ensure the stability and accuracy of alkaline phosphatase during chemiluminescence detection. In addition, a large amount of metal ions including zinc ions, sodium ions, magnesium ions or calcium ions are added in the form of inorganic salts, which not only provides an electrolyte environment for the chemiluminescence of alkaline phosphatase magnetic particles, but also reduces the binding force between foreign proteins and antibodies.

[0029] Regarding the selection of surfactants, nonionic surfactants and / or imidazoline-type zwitterionic surfactants can be selected, and cationic surfactants (such as Gemini) are particularly avoided; wherein, nonionic surfactants include: sorbitol polyoxyethylene ether laurate, sorbitol polyoxyethylene ether oleate, lauryl alcohol polyoxyethylene ether (such as Brij35), alkylphenol polyoxyethylene ether (such as APEO or OP series) and / or Tween 20. Through screening experiments, it was found that Tween 20 (Tween 20) and Brij35 can effectively reduce the physical adsorption between proteins and reaction cups and needles, and the effect is relatively more significant. In addition, the imidazole-based surfactants are screened from the amphoteric surfactants. The imidazole-based surfactants are cross-products of imidazole functionalized ions and the field of surfactants. In its molecular structure, the hydrophobic long chain is connected to one or two N atoms of the imidazole ring and the imidazole ring is used as the hydrophilic cationic head group. The present invention screens out 1-sulfopropyl-3-dodecyl imidazole inner salt (abbreviated as C 12 imSP), C 12 As an amphoteric imidazole ionic liquid surfactant, imSP has low toxicity and good biodegradability. It contains two molecular structures, sulfonic group and imidazole ring group. Among them, the hydrophilic group represented by the sulfonic group can carry a negative charge in an alkaline environment (it is necessary to avoid the osmotic pressure and ion shielding effect formed by a high salt environment), which ensures that the surfactant can show good surface activity in an alkaline medium; the ionic liquid with the imidazole ring as the core cation is relatively not easy to hydrolyze and has better thermal stability. The present invention has been experimentally verified that C 12 imSP can effectively clean up nonspecific impurities without destroying the binding of antigen and antibody. 12 imSP may be connected to the surface molecules of alkaline phosphatase through hydrogen bonds, protecting the native conformation of the protein and avoiding protein denaturation due to the use of surfactants, thereby improving the stability of alkaline phosphatase.

[0030] In order to avoid the adverse effects of foam generated during the cleaning process on the cleaning effect and the test results, a defoaming agent can be appropriately added. The defoaming agent of the present invention is preferably a polyether defoaming agent. Compared with the silicone oil defoaming agent, the polyether defoaming agent can maintain a good defoaming effect at a lower concentration, thereby reducing the production cost. In addition, the polyether defoaming agent does not generate harmful gases and harmful substances during use, and is easy to decompose, meeting environmental protection standards.

[0031] Regarding preservatives, the preservative Proclin 300 mentioned in CN111607465A is a single preservative and cannot achieve a good preservative effect. The present invention prefers the single preservative Krovin 500, which is a 40% aqueous solution of pyridine sodium salt as an active ingredient. Compared with other commonly used preservatives such as isothiazolinone, formaldehyde-releasing type, benzimidazole, substituted aromatic hydrocarbons, etc., Krovin 100 / 300 / 500 series preservatives do not release formaldehyde, do not irritate the skin, have thermal stability, acid-base stability, a broad antibacterial spectrum, and are environmentally friendly and safe due to being degradable. The production cost is equivalent to or slightly lower than PC-950 and BND-10. 10. Although the production cost is relatively high (compared to organic acids and synthetic preservatives such as sodium benzoate and EDTA), the Krovin series preservatives have the common characteristics of good compatibility, good chemical stability, low toxicity and safety. In particular, the present invention can use Krovin300 (pH value is 2-9.5) or Krovin500 (pH value is 5-11) alone, breaking the technical limitation that a single component preservative can achieve a better preservative effect, and can also be promoted and applied to products such as reference substances, coating solutions, and sealing solutions.

[0032] In summary, the present invention provides a cleaning solution, a preparation method and its application in alkaline phosphatase magnetic microparticle chemiluminescence. The formula of the cleaning solution includes the optimization and screening of the buffer system, metal ions, surfactants and preservatives. The pH value is preferably in the slightly lower range of 8 to 9, which reduces the adverse effects on the protein and specific binding in the sample to be tested while satisfying the alkaline phosphatase activity. In particular, the synergistic effect of using nonionic surfactants and zwitterionic surfactants at the same time, C is preferably selected from the zwitterionic surfactants. 12 imSP, simple preparation, reducing reagent costs. The use restriction of a single component is broken in the screening of preservatives, and the Krovin series is preferred, which has a wide antibacterial spectrum and further improves the stability of the cleaning solution. The cleaning solution optimized by the present invention can be used on a fully automatic biochemical analyzer, has a high degree of automation, and the mother liquor concentrated configuration makes storage and transportation more convenient. The cleaning solution optimized by the present invention can also improve the detection sensitivity, specificity and accuracy of the chemiluminescence of alkaline phosphatase magnetic microparticles, with a larger detection range and better stability. DETAILED DESCRIPTION

[0033] In the following detailed description, for the convenience of explanation, many specific details are set forth to provide a comprehensive understanding of the embodiments of the present disclosure. The following embodiments are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention.

[0034] The experimental methods used in the following examples are conventional methods unless otherwise specified, and the materials and reagents used are all commercially available unless otherwise specified. Among them, AMPPD substrate was purchased from Philips, and Tween 20, Brij35, salts containing metal ions such as zinc ions, magnesium ions, and calcium ions, and other common reagents were purchased from Sigma; C 12 imSP was synthesized by the laboratory; the procalcitonin chemiluminescence kit was a reagent developed by the laboratory; and the fully automatic chemiluminescence analyzer SMART 500H was purchased from Cosmay Instruments. However, it is obvious that one or more embodiments can be implemented without these specific details. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer or the product instructions.

[0035] C 12 Laboratory preparation process of imSP: C was prepared from imidazole, acrylonitrile, 1-bromododecane and propane sultone. 12 imSP, dissolve imidazole in methanol at 50°C and react with acrylonitrile for 8h to obtain ethyl imidazole, then react with 1-bromododecane in isopropanol solvent at 80°C for 18h, remove isopropanol by rotary evaporation, mix the residue with NaOH solution at a mass ratio of 15% and stir, then extract with chloroform to obtain 1-dodecyl imidazole, then react with propane sultone in ethanol / water solvent at 75°C and pH 8-9 for 8h to obtain the target product C 12 imSP, through physical and chemical properties and indicators such as washing, emulsification, dispersion and foaming performance, all meet the standards.

[0036] The present invention provides a preferred formula of a cleaning solution, which includes optimized screening of a buffer system, metal ions, surfactants, preservatives and defoamers, and further reduces the pH value to 8-9, thereby ultimately improving the cleaning ability and detection sensitivity while ensuring the activity of alkaline phosphatase. The specific embodiments of the present invention are described below:

[0037] Example 1: Cleaning liquid formula 1

[0038] The cleaning solution formula is: 50mM Tris, 100mM calcium acetate, 0.01% zinc chloride, 40g / L sodium chloride, 0.5% Tween 20, 0.05% Brij35, 0.1% C 12 imSP, 0.05% Krovin100, 0.5% Krovin300, 1% polyether defoamer. Add the above raw materials to purified water at a concentration ratio or concentration of 10 times the mother liquor, stir evenly, adjust the pH to 8.8, and filter with a 0.22um filter membrane to prepare a mother liquor for storage, store at 4°C after aliquoting, and dilute with purified water before use.

[0039] Example 2: Cleaning liquid formula 2

[0040] The cleaning solution formula is: 100mM Tris, 100mM magnesium sulfate, 0.1% zinc chloride, 200g / L sodium chloride, 0.05% Tween 20, 1% Brij35, 1% C 12 imSP, 0.5% Krovin300, 0.5% polyether defoamer. Add the above raw materials to purified water at a concentration ratio or concentration of 15 times the mother liquor, stir evenly, adjust the pH to 8.5, and filter with a 0.22um filter membrane to prepare a mother liquor for storage, store at 4°C after aliquoting, and dilute with purified water before use.

[0041] Example 3: Cleaning liquid formula 3

[0042] The cleaning solution formula is: 200mM Tris, 5mM magnesium chloride, 10mM calcium chloride, 0.2% zinc chloride, 100g / L sodium chloride, 1% Tween 20, 0.5% Brij35, 0.5% C 12 imSP, 1% Krovin500, 0.1% polyether defoamer, add the above raw materials into purified water according to the concentration ratio or concentration of 20 times the mother liquor, stir evenly, adjust the pH to 8.3, and filter with a 0.22um filter membrane to prepare a mother liquor for storage. Store at 4°C after aliquoting and dilute with purified water before use.

[0043] Example 4: Cleaning solution control

[0044] The cleaning solution is prepared according to the optimal formula disclosed in CN115340910B, specifically including 2% Tris, 10% NaCl, 5% magnesium chloride, 2% Tween 20, 1% S9, 1% PC-950, 1% BND-10, 0.1% Gemini surfactant and 0.1% polyether defoamer. The above raw materials are added to purified water at a concentration ratio or concentration of 20 times the mother liquor, and the pH is adjusted to 9.5 after stirring evenly, and filtered with a 0.22um filter membrane to prepare a mother liquor for storage. After packaging, it is stored at 4°C and diluted with purified water before use.

[0045] Example 5: Performance Evaluation

[0046] The cleaning solutions prepared in Examples 1 to 4 were used in combination with a procalcitonin chemiluminescence kit for detection and comparison. The specific detection contents included blank value determination, linearity determination, precision determination, and stability determination. The specific detection process and results are as follows.

[0047] Determination of blank value: The following experiment was performed according to the normal operating procedure recommended by the fully automatic chemiluminescence analyzer: 30uL sample, 50uL magnetic beads, and 50uL ALP marker were added in sequence and mixed evenly, and the mixture was incubated at 37°C for 15min, and then the cleaning solutions described in Examples 1 to 4 were used for 4 cleanings, and then 200uL AMPPD substrate was added, and the mixture was incubated at 37°C for 5min, and the number of photons was measured. The sample was the same blank calibrator (the content of procalcitonin was 0ng / mL), and the measurement was repeated 20 times to observe the change in the coefficient of variation (coefficient of variation CV = (standard deviation SD / mean value M) × 100%). The test results are shown in Table 1 below.

[0048] Table 1 Blank value test results

[0049] serial number Example 1 Example 2 Example 3 Comparison example 1 2332 2676 2248 2934 2 3552 6267 2497 5960 3 4881 4540 2464 3967 4 3056 5179 3748 2442 5 3191 2486 3052 4347 6 4218 2712 3118 2451 7 3459 3823 2781 5587 8 5411 3092 2589 3777 9 1944 5675 2642 2165 10 4138 3207 2341 4203 11 2418 2489 2856 2485 12 1984 3485 2478 2478 13 3975 2487 3074 3954 14 3974 3018 3014 5104 15 4187 2748 2894 2854 16 4789 4859 3514 2489 17 4781 6214 3120 4101 18 4105 2485 3102 2895 19 4411 3847 2514 4984 20 4101 4891 2145 4101 M 3745 3809 2810 3664 SD 990 1302 417 1154 CV 26.4％ 34.2％ 14.8％ 31.5％

[0050] It can be seen from Table 1 that with the same reagents and the same sample to be tested, the blank value of the cleaning solution prepared by Example 3 of the present invention is relatively low, and the coefficient of variation of Example 3 is also significantly lower than that of the other three groups of examples.

[0051] Linearity evaluation: The following experiment was conducted according to the normal operating procedures recommended by the fully automatic chemiluminescence analyzer: 30uL of sample, 50uL of magnetic beads, and 50uL of ALP marker were added in sequence and mixed evenly. After incubation at 37°C for 15min, the samples were washed 4 times using the cleaning solutions described in Examples 1 to 4, and then 200uL of AMPPD substrate was added. After incubation at 37°C for 5min, the number of photons was determined. The samples were the same blank control and procalcitonin calibrator with a concentration in the range of 0.02 to 100ng / ml. The linear correlation coefficients r of the test results using different cleaning solutions are shown in Table 2 below.

[0052] Table 2 Linearity determination results

[0053] Concentration gradient (ng / mL) Example 1 Example 2 Example 3 Example 4 0 3145 3966 2748 3783 0.02 4977 9047 5912 6631 0.2 32859 36054 36786 33655 2 303463 346647 359135 304278 20 3522573 3913177 4036694 3667318 200 17452144 21188331 22403741 18847968 r 0.9948 0.9964 0.9968 0.9955

[0054] As can be seen from Table 2: the cleaning solutions prepared in Examples 1 to 4 are relatively stable in the detection range of 0 to 100 ng / ml for the same blank value and the detection signal value of the sample to be tested, and there is a strong linear correlation. The detection results meet the linear standard, and the correlation coefficient r detected in Example 3 is relatively the highest.

[0055] Precision evaluation: The following experiment was conducted according to the normal operating procedures recommended by the fully automatic chemiluminescence analyzer: 30uL sample, 50uL magnetic beads, and 50uL ALP marker were added in sequence and mixed evenly; the mixture was incubated at 37°C for 15min, and then the cleaning solutions described in Examples 1 to 4 were used for 4 washes, and then 200uL AMPPD substrate was added; the mixture was incubated at 37°C for 5min, and the number of photons was determined. The samples were mixed clinical serum samples, and three concentrations of low, medium, and high were quantitatively set. Each test was repeated 10 times. The changes in the coefficient of variation CV detected using different cleaning solutions are shown in Table 3 below.

[0056] Table 3 Precision determination

[0057]

[0058] As can be seen from Table 3, compared with the control, the coefficient of variation CV of the three cleaning solution formulas of the present invention for the detection of samples of low, medium and high concentrations is less than 10%, and the difference between the test results is small. It can be seen that the stability and repeatability of the detection of different concentrations are relatively good, and the test results are stable and reliable, which can more accurately reflect the true concentration of the sample. In particular, the coefficient of variation CV of the detection of samples of low, medium and high concentrations by the cleaning solution described in Example 3 of the present invention is less than 5%, and the difference between the test results is very small and almost negligible. Therefore, it has extremely high stability and accuracy, greatly reducing the risk of misjudgment or missed judgment due to detection errors, and can be selected as a more ideal cleaning solution formula.

[0059] Thermal acceleration stability evaluation: The four diluted cleaning solutions prepared in Examples 1 to 4 were placed at 37°C for 3 days, 7 days, and 14 days, respectively. Then, referring to the above-mentioned precision evaluation method, the mixed clinical serum samples with three quantitative settings of low, medium, and high concentrations were tested. Each test was repeated 3 times. The change in the coefficient of variation CV after the thermal acceleration experiment of the cleaning solution is shown in Table 4 below.

[0060] Table 4 Thermal acceleration stability results

[0061]

[0062] It can be seen from Table 4 that the three cleaning solution formulas prepared in Examples 1 to 3 have good thermal stability, and the coefficient of variation CV is less than 10%, and the detection results corresponding to the cleaning solutions are relatively stable and reliable; especially when the cleaning solution described in Example 3 of the present invention is used, compared with the control group, the coefficient of variation CV of the detection of low, medium and high concentration samples after the thermal acceleration experiment using the cleaning solution described in Example 3 is still maintained at less than 5%, which shows that the formula has good stability and is convenient for long-term storage. If it is prepared into a 10-20 times concentrated mother liquor, the stability can also meet the requirements in theory. In addition, the present invention optimizes the formula of the preservative. It can be seen that especially in Examples 2 to 3, the use of a single-ingredient preservative can also ensure the stability of the reagent, and a significant beneficial effect has been achieved.

[0063] The specific embodiments of the present invention have been described in detail so that those skilled in the art can easily understand. However, according to all the descriptions disclosed, different modifications or replacements can be made to those details, and these changes are within the scope of protection of the present invention. The full scope of the present invention is given by the attached claims and any equivalents thereof.

Claims

1. A cleaning liquid, characterized in that: Used for the detection of chemiluminescence of alkaline phosphatase magnetic microparticles, the cleaning solution comprises a buffer solution, metal ions, a surfactant and a preservative, and the pH value is 8 to 9; The buffer is Tris buffer with a working concentration of 50-200 mM; The metal ions include at least zinc ions and sodium ions, which are derived from zinc chloride and sodium chloride, respectively; the metal ions also include at least magnesium ions and / or calcium ions, wherein the magnesium ions are derived from soluble salts containing magnesium ions, such as magnesium chloride and / or magnesium sulfate; the calcium ions are derived from soluble salts containing calcium ions, such as calcium chloride and / or calcium acetate; The surfactant comprises both a nonionic surfactant and a zwitterionic surfactant, wherein the zwitterionic surfactant is an imidazoline-type structure; The preservative is a Krovin series preservative.

2. The cleaning liquid according to claim 1, characterized in that: The working concentration of the zinc ion is 0.01-0.2 mM, the working concentration of the sodium ion is 40 g / L-200 g / L; the working concentration of the magnesium ion or the calcium ion is 1-100 mM; the nonionic surfactants are Tween 20 and Brij35, both of which have a working concentration of 0.05%-1%; the working concentration of the zwitterionic surfactant is 0.1%-1%; the working concentration of the preservative is 0.05%-1%; and a defoamer may also be included, which is a polyether defoamer with a working concentration of 0.01-0.1%.

3. The cleaning solution according to claim 1, characterized in that: The pH value is 8.3-8.8; the zwitterionic surfactant is 1-sulfopropyl-3-dodecyl imidazole inner salt.

4. The cleaning liquid according to claim 1, characterized in that: The total working concentration of the magnesium ions and / or the calcium ions does not exceed 100 mM; the total mass concentration of the nonionic surfactant does not exceed 1.5%.

5. The cleaning liquid according to claim 1, characterized in that: The Krovin series preservatives include Krovin100, Krovin300 and Krovin500, and any one of the Krovin series preservatives can be selected and used alone.

6. The cleaning liquid according to claim 3, characterized in that: The 1-sulfopropyl-3-dodecyl imidazole inner salt is prepared by itself using imidazole, acrylonitrile, 1-bromododecane and propane sultone as raw materials.

7. The cleaning liquid according to claim 6, characterized in that: The preparation method of the 1-sulfopropyl-3-dodecyl imidazole inner salt is as follows: imidazole is dissolved in methanol at 40-60° C., acrylonitrile is added and reacted for 6-10 hours to obtain nitrile ethyl imidazole; the nitrile ethyl imidazole and 1-bromododecane are reacted in an isopropanol solvent at 70-90° C. for 16-20 hours, the isopropanol is removed by rotary evaporation, the obtained residue is stirred with a NaOH solution at a mass ratio of 10-20%, and then mixed to obtain a mixed solution; the mixed solution is extracted with chloroform to obtain 1-dodecyl imidazole; the 1-dodecyl imidazole is placed in an ethanol / water solvent at 70-80° C. and a pH value of 8-9, propane sultone is added and reacted for 6-10 hours, and the reaction product includes the target product 1-sulfopropyl-3-dodecyl imidazole inner salt.

8. The cleaning liquid according to claim 6, characterized in that: According to the cleaning solution according to any one of claims 1 to 5, the working concentration is converted into a mother liquor concentrated 10 to 20 times for preparation or storage, and diluted with purified water to the working concentration when used.

9. A method for preparing a cleaning solution, characterized in that: According to any one of claims 1 to 7, the working concentration is converted into a 10-20 times concentrated ratio and added to purified water, the pH value is adjusted after stirring evenly, and filtered with a 0.22 um filter membrane to obtain the mother liquor of the cleaning solution, which is packaged and stored at 4° C. for use.

10. The cleaning solution as claimed in any one of claims 1 to 8, the preparation method of the cleaning solution as claimed in claim 9, and use thereof in the preparation of products related to alkaline phosphatase chemiluminescence detection.

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